EP3592392B1 - Biomatériaux à modification fasl ayant une fonction immunomodulatrice - Google Patents

Biomatériaux à modification fasl ayant une fonction immunomodulatrice Download PDF

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EP3592392B1
EP3592392B1 EP18764623.7A EP18764623A EP3592392B1 EP 3592392 B1 EP3592392 B1 EP 3592392B1 EP 18764623 A EP18764623 A EP 18764623A EP 3592392 B1 EP3592392 B1 EP 3592392B1
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Prior art keywords
fasl
biomaterial
cells
graft
subject
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German (de)
English (en)
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EP3592392A4 (fr
EP3592392A1 (fr
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Haval Shirwan
Andres J. Garcia
Esma S. Yolcu
Hong Zhao
Devon Headen
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Georgia Tech Research Corp
University of Louisville Research Foundation ULRF
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Georgia Tech Research Institute
Georgia Tech Research Corp
University of Louisville Research Foundation ULRF
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Priority to RS20230876A priority Critical patent/RS64754B1/sr
Priority to SI201831008T priority patent/SI3592392T1/sl
Priority to HRP20231297TT priority patent/HRP20231297T1/hr
Priority to SM20230437T priority patent/SMT202300437T1/it
Priority to EP23196606.0A priority patent/EP4299749B1/fr
Publication of EP3592392A1 publication Critical patent/EP3592392A1/fr
Publication of EP3592392A4 publication Critical patent/EP3592392A4/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/178—Lectin superfamily, e.g. selectins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/4353—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems
    • A61K31/436—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom ortho- or peri-condensed with heterocyclic ring systems the heterocyclic ring system containing a six-membered ring having oxygen as a ring hetero atom, e.g. rapamycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6903—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being semi-solid, e.g. an ointment, a gel, a hydrogel or a solidifying gel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M3/00—Tissue, human, animal or plant cell, or virus culture apparatus
    • C12M3/02—Tissue, human, animal or plant cell, or virus culture apparatus with means providing suspensions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells

Definitions

  • FasL-engineered biomaterials and the biomaterials for use such as for immunomodulation, such as for treating autoimmune diseases including Type 1 diabetes and preventing or reducing the risks of graft rejection.
  • transplantation of foreign cells has become an important and effective therapeutic alternative for patients with certain diseases.
  • foreign cells such as bone marrow and stem cells
  • tissues such as pancreatic islets
  • organs such as kidneys, hearts, livers
  • transplantation of foreign grafts between genetically different patients is limited by the ability to control the immunological recognition and rejection of the graft by the recipient.
  • autografts where the graft cells are derived from the patient's own tissue, for example, by induced pluripotency
  • the efficacy of the transplantation will depend on controlling the autoimmune response to the grafts.
  • bone marrow (BM) transplantation has been viewed as an extraordinarily promising treatment for hematopoietic and autoimmune disorders and for certain cancers.
  • One obstacle to bone marrow transplantation is the possibility of rejection of the transplanted tissue, mediated by the host's T cells and NK cells.
  • Graft-versus-host-disease (GvHD) is another possible adverse consequence of bone marrow transplantation.
  • Donor T cells in the transplanted tissue can mount an immune response against the host's vital organs, often leading to death of the host.
  • Host-versus-graft reactions and GvHD therefore limit the clinical use of bone marrow transplantation, which might otherwise be widely used to treat various diseases and to prevent foreign graft rejection.
  • Type 1 diabetes is an autoimmune disease characterized by loss of insulin-producing ⁇ -cell mass, and thereby glycemic control, due to a coordinated immune response against ⁇ -cell specific antigens requiring CD4+ T cells.
  • Restoration of ⁇ -cell mass through allogeneic islet transplantation is currently the preferred clinical intervention to improve glycemic control in patients with severe glycemic instability.
  • Even with autologous beta cell products, controlling the immune response to the autologous cells will remain important to therapeutic efficacy. Longevity of allogeneic grafts is limited not only by host immune responses, but also by secondary graft failure due to toxic effects of chronic immunosuppression required to control rejection.
  • Immunosuppressive pharmacological agents are a mainstay of regimens for the control of allograft rejection. Although such drugs are effective in reducing the severity of rejection episodes, they are nonspecific and fail to create a state of permanent graft-specific tolerance. Continuous exposure of the recipient to these immunosuppressive agents is therefore associated with a significantly increased risk of opportunistic infections and malignancies. Additionally, these nonspecific immunosuppressive agents can induce serious and undesirable side effects in the host. These adverse effects often outweigh the benefits for patients with diseases in which the body identifies certain parts of itself as "foreign" and launches an adaptive immune attack that results in autoimmunity, such as is observed in type 1 diabetes, arthritis, lupus, and multiple sclerosis.
  • compositions and methods useful for effecting immunomodulation such as for preventing or reducing the risks of rejection of cellular or tissue grafts and/or the treatment of Type I diabetes.
  • compositions and methods useful for inducing immune tolerance are also is a need, therefore, for compositions and methods useful for inducing immune tolerance.
  • the invention is the FasL-engineered biomaterials as defined in claim 1, as well as the biomaterial as defined in claim 10 for use in inducing immune tolerance, and a method of making such FasL-engineered biomaterials as defined in claim 21.
  • biomaterial engineered to display a FasL protein wherein a hydrogel is engineered to display FasL proteins, and wherein the hydrogel comprises a chimeric FasL protein comprising a FasL moiety and a streptavidin or avidin moiety conjugated via biotin to the hydrogel.
  • the hydrogel is a polyethylene glycol (PEG) microgel engineered to display a biotin moiety.
  • PEG polyethylene glycol
  • the hydrogels may comprise biotin moieties conjugated to SA-FasL moieties.
  • the FasL moiety may be a matrix metalloproteinase resistant FasL protein.
  • the biomaterial may comprise an immunosuppressive drug, such as rapamycin.
  • the FasL-engineered hydrogels may further comprise an immunosuppressive drug, such as rapamycin. FasL-engineered biomaterials or hydrogels that further comprise an immunosuppressive drug may provide controlled release of the drug.
  • the biomaterial may comprise a graft cell, such as PBMCs, bone marrow cells, hematopoietic stem cells, stem cells, mesenchymal stem cells, dendritic cells, dendritic cells pulsed with autoantigens, human beta cell products, and splenocytes.
  • a graft cell such as PBMCs, bone marrow cells, hematopoietic stem cells, stem cells, mesenchymal stem cells, dendritic cells, dendritic cells pulsed with autoantigens, human beta cell products, and splenocytes.
  • the graft cell may be encapsulated in the biomaterial.
  • a biomaterial according to the invention for use in inducing immune tolerance in a subject in need thereof.
  • the administering is by transplantation.
  • the subject may be a human, a non-human primate, a pig, a dog, a cat, a cow, a sheep, a horse, a rabbit, a mouse, or a rat.
  • the subject is in need of immune tolerance to a graft cell, such as PBMCs, bone marrow cells, hematopoietic stem cells, stem cells, mesenchymal stem cells, dendritic cells, dendritic cells pulsed with autoantigens, human beta cell products, and splenocytes.
  • a graft cell such as PBMCs, bone marrow cells, hematopoietic stem cells, stem cells, mesenchymal stem cells, dendritic cells, dendritic cells pulsed with autoantigens, human beta cell products, and splenocytes.
  • the biomaterial may be for use for preventing or reducing the risks of rejection of cellular or tissue grafts and/or the treatment of type 1 diabetes.
  • the biomaterial is for use in a subject, where the subject is in need of immune tolerance to a graft cell, such as PBMCs, bone marrow cells, hematopoietic stem cells, stem cells, mesenchymal stem cells, dendritic cells, dendritic cells pulsed with autoantigens, human beta cell products, and splenocytes.
  • a graft cell such as PBMCs, bone marrow cells, hematopoietic stem cells, stem cells, mesenchymal stem cells, dendritic cells, dendritic cells pulsed with autoantigens, human beta cell products, and splenocytes.
  • the biomaterial comprises the graft cell.
  • the graft cell is encapsulated in the biomaterial.
  • the subject is in need of treatment for type 1 diabetes, and the biomaterial may be for use for administering pancreatic islet cells to the subject.
  • the subject is in need of treatment or prevention of allograft rejection, and the biomaterial may be for use for administering to the subject cells from an allograft donor.
  • the subject is in need of treatment or prevention of xenograft rejection, and the biomaterial may be for use for administering to the subject cells from a xenograft donor.
  • the xenograft donor is a human, a non-human primate, a pig, a dog, a cat, a cow, a sheep, a horse, a rabbit, a mouse, or a rat.
  • the subject is in need of treatment or prevention of autograft rejection, and optionally the subject is administered autologous graft cells.
  • the autologous graft cells may be obtained by induced pluripotency.
  • the subject is in need of treatment or prevention of autoimmunity, and optionally the subject is administered an autoantigen presented on a cell selected from (i) a cell expressing the autoantigen (ii) a cell decorated with the autoantigen and (iii) a dendritic cell pulsed with the autoantigen.
  • the FasL-engineered biomaterials as defined in claim 1 are useful, for example, for inducing immune tolerance or immunosuppression, such as may be desired in the context of treating autoimmune disease or treating or preventing graft rejection.
  • FasL-mediated apoptosis is critical to the induction of self-tolerance and maintenance as deficiency in Fas or FasL is associated with massive autoimmunity both in humans and in rodents. This suggests that there are no compensatory mechanisms for this pathway, further emphasizing its importance as a target for immunomodulation.
  • FasL-engineered biomaterials as defined in claim 1 provide controlled loading, presentation, and retention of FasL protein at target sites in vivo, and are effective for immunomodulation.
  • the FasL-engineered biomaterials may be co-administered with a graft ( e.g ., with graft cells or graft tissue), and induce immune tolerance to the graft.
  • the biomaterials of the invention may achieve long-term, specific immunosuppression at the graft site, avoiding the toxicity associated with non-specific, systemic pharmacologic immunosuppressants.
  • FasL This is a unique advantage over gene therapy, because uncontrolled, continuous expression of FasL, which possesses pleiotropic functions and different modes of expression that may be differentially regulated by the target tissues (membrane bound or soluble), may have unintended consequences. Indeed, ectopic expression of FasL using gene therapy for immunomodulation in transplantation settings has resulted in mixed and opposing outcomes with some studies showing a detrimental impact of FasL expression on graft survival.
  • the localized and sustained presentation of FasL as described herein overcomes complications associated with ectopic expression of wild-type FasL in target tissues using gene therapy. This localized immunomodulation concept also limits potential toxicities associated with agonistic antibodies against Fas for immunomodulation.
  • the FasL-engineered biomaterials of the invention provide the flexibility of an off-the-shelf product for wider clinical applications, as these immunomodulatory materials can be prepared at the time of transplantation and simply co-mixed with graft cells (such as islets) for delivery without the need of encapsulating the graft cells or manipulating graft cells to present proteins.
  • graft cells such as islets
  • CD8 + and CD4 + T effector cells play a critical role in the initiation and perpetuation of various autoimmune diseases, including type 1 diabetes, rheumatoid arthritis, lupus, multiple sclerosis, and in foreign graft rejection, including rejection of allogeneic and xenogeneic grafts.
  • T effector cells therefore, represent an important target for immune modulation to prevent and treat these diseases.
  • T effector (Teff) cells are kept in check by another class of T cells, designated as T regulatory (Treg) cells. Treg cells, similar to Teff cells, follow the inflammatory cues and infiltrate into rejecting grafts.
  • administering includes directly administering to another, self-administering, and prescribing or directing the administration of an agent as disclosed herein.
  • administering encompasses all suitable means of providing a substance to a patient. Common routes include oral, sublingual, transmucosal, transdermal, rectal, vaginal, subcutaneous, intramuscular, intravenous, intra-arterial, intrathecal, via catheter, via implant etc.
  • Patient or “subject” as used herein includes any mammal. In some embodiments, the patient is human.
  • the phrases "effective amount” and “therapeutically effective amount” mean that dosage or plasma concentration in a subject, respectively, that provides the specific pharmacological effect for which the active agent is administered in a subject in need of such treatment. It is emphasized that an effective amount of an active agent will not always be effective in treating the conditions/diseases described herein, even though such dosage is deemed to be an effective amount by those of skill in the art.
  • composition refers to one or more active agents formulated with a pharmaceutically acceptable carrier, excipient or diluent.
  • phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in vivo without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • the FasL-engineered biomaterials of the invention are biomaterials engineered to display a FasL protein.
  • FasL refers to the Fas ligand.
  • FasL moiety means at least the apoptosis-inducing moiety of FasL.
  • the FasL moiety comprises or consists of the extracellular domain of FasL.
  • the FasL moiety comprises or consists of a matrix metalloproteinase (MMP) resistant FasL protein.
  • MMP matrix metalloproteinase
  • the matrix metalloproteinase (MMP) resistant FasL protein is a form of FasL in which the extracellular domain of FasL lacks MMP sensitive sites. See Yolcu et al., Immunity 17: 795-808 (2002 ).
  • biomaterials are engineered to display FasL by conjugation via biotin to a hydrogel as defined in claim 1, and controlled release technologies can be used.
  • the FasL protein is displayed on the biomaterial via biotin/avidin or biotin/streptavidin (SA) binding.
  • SA biotin/avidin or biotin/streptavidin
  • a hydrogel is biotinylated and bound to a FasL-streptavidin conjugate (or a chimeric protein comprising a FasL protein and a streptavidin or avidin moiety) via streptavidin-biotin binding.
  • SA-FasL tethered to biotinylated hydrogels retains potent apoptotic activity.
  • the quantity of bioactive SA-FasL delivered to a subject can be easily controlled using the FasL biomaterials described herein.
  • biotin has an extremely high affinity for both SA (10 13 M -1 ) and avidin (10 15 M -1 ).
  • both SA and avidin are tetrameric polypeptides that each bind four molecules of biotin. Conjugates comprising SA or avidin therefore have a tendency to form tetramers and higher structures, and can form complexes with multiple biotin-containing moieties.
  • biotin includes biotin-containing moieties that are able to bind to surfaces, such as cell surfaces, such as NHS-biotin and EZ-Link TM Sulfo-NHS-LC-Biotin (Pierce). Biotin and protein-reactive forms of biotin are available commercially.
  • SA or avidin fragments which retain substantial binding activity for biotin, such as at least 50% or more of the binding affinity of native SA or avidin, respectively, also may be used.
  • Such fragments include "core streptavidin” ("CSA"), a truncated version of the full-length streptavidin polypeptide which may include streptavidin residues 13-138, 14-138, 13-139 or 14-139. See, e.g., Pahler et al., J. Biol. Chem., 262: 13933-37 (1987 ).
  • Other truncated forms of streptavidin and avidin that retain strong binding to biotin also may be used. See, e.g. Sano et al., J Biol Chem.
  • mutants with reduced immunogenicity such as mutants mutated by site-directed mutagenesis to remove potential T cell epitopes or lymphocyte epitopes
  • mutants of avidin and core forms of avidin which retain substantial biotin binding activity or increased biotin binding activity also may be used.
  • Hiller et al. J Biochem, 278: 573-85 (1991 ); and Livnah et al., Proc Natl Acad Sci USA 90: 5076-80 (1993 ).
  • the terms "avidin” and “streptavidin” encompass fragments, mutants and core forms of these molecules.
  • Avidin and streptavidin are available from commercial suppliers. Moreover, the nucleic acid sequences encoding streptavidin and avidin and the streptavidin and avidin amino acid sequences are known. See, e.g., GenBank Accession Nos. X65082; X03591; NM_205320; X05343; Z21611; and Z21554.
  • the biomaterial is a hydrogel.
  • hydrogel refers to a water swollen polymer material, e.g ., water-swollen polymer networks, with dimensions much larger than a cell (such as > 500 ⁇ m).
  • microgel refers to a hydrogel with smaller dimensions (such as on the order of 10s or 100s of ⁇ m).
  • the hydrogel may be any pharmaceutically acceptable hydrogel that is suitable for administration into the target subject.
  • a hydrogel typically is formed when an organic polymer (natural or synthetic) is crosslinked via covalent, ionic, or hydrogen bonds to create a three-dimensional open-lattice structure which entraps water molecules to form a gel.
  • Examples of materials which can be used to form a hydrogel include macromer-based materials (including PEG macromers) assembled using different crosslinking methods (such as Michael-type addition, thiol-ene, click reactions, etc), polysaccharides (such as alginate), polyphosphazines, and polyacrylates, or block copolymers such as Pluronics TM or Tetronics TM , polyethylene oxide-polypropylene glycol block copolymers which are crosslinked by temperature, free radical polymerization, click reactions or pH, respectively.
  • macromer-based materials including PEG macromers
  • crosslinking methods such as Michael-type addition, thiol-ene, click reactions, etc
  • polysaccharides such as alginate
  • polyphosphazines such as alginate
  • polyphosphazines such as alginate
  • polyacrylates such as Pluronics TM or Tetronics TM
  • Pluronics TM or Tetronics TM polyethylene oxide-polyprop
  • the biomaterial is a polyethylene glycol (PEG) hydrogel or microgel.
  • the hydrogel may be synthesized from maleimide-terminated 4-arm poly(ethylene) glycol (PEG-4MAL) macromers, such as by microfluidics polymerization. See Headen et al., Advanced Materials, 26:3003-3008 (2014 ).
  • PEG-4MAL platform enables stoichiometric, covalent incorporation of thiol-containing molecules, and provides improved crosslinking efficiency for formation of structurally defined hydrogels. See Phelps et al., Advanced Materials, 24: 64-70, 62 (2012 ).
  • PEG-4MAL exhibits minimal toxicity in vivo, and it is rapidly excreted in the urine, important considerations for clinical applications.
  • Biotinylated hydrogels or microgels can be produced by reacting biotin-PEG-thiol with PEG-4MAL macromer, and generating 150 ⁇ m diameter microgels crosslinked with dithiothreitol (DTT) via microfluidics polymerization. See, e.g., FIG. 1A .
  • the resulting microgels display covalently-tethered biotin capable of capturing SA with high affinity. See, e.g., FIG. 1B .
  • the biomaterial may comprise or be formulated with ( e.g ., admixed with or blended with) an additional therapeutic agent, such as an immunosupressant.
  • an additional therapeutic agent such as an immunosupressant.
  • suitable immunosuppressive drugs include rapamycin, cyclophosamide busulfan, fludarabine, methotrexate, sulfasalazine, hydroxychloroquine, azathioprine, tocilizumab, etanercept, adalimumab, anakinra, abatacept, rituximab, certolizumab, golimumab, cyclosporine, dexamethasone, methylprednisolone, predinisone, tacrolimus and triamcinolone.
  • the immunosuppressive drug is rapamycin.
  • the biomaterial of the invention may be applied in methods of effecting immunomodulation comprising administering to a subject in need thereof a FasL-engineered biomaterial as described herein.
  • the method may be for preventing or reducing the risks of rejection of cellular or tissue grafts and/or the treatment of Type I diabetes.
  • the FasL biomaterials described herein are useful for inducing immunosuppression.
  • the biomaterials may be used in methods of inducing immunosuppression in a subject in need thereof comprising administering to the subject a FasL biomaterial in an amount effective to induce immune tolerance.
  • the FasL biomaterials described herein also are useful for inducing specific immune tolerance.
  • administering a FasL biomaterial along with a graft may induce specific immune tolerance to the graft cell.
  • a graft e.g., a graft cell
  • biomaterials for use in inducing immune tolerance in a subject in need thereof whereby the subject is administered a graft cell FasL biomaterial in an amount effective to induce immune tolerance to the graft cell.
  • graft cell refers to a donor cell (or tissue or organ comprising a cell), that is administered to a subject in need thereof.
  • Types of graft cells include islet cells (e.g ., pancreatic islet cells), splenocytes, PBMCs, bone marrow cells, mesenchymal stem cells, hematopoietic stem cells, stem cells, induced pluripotent stem cells, human beta cell products, hepatocytes, dendritic cells, macrophages, endothelial cells, cardiac myocytes, and vascular cells, and immune cells, including T cells, etc., depending on the condition being treated.
  • the FasL hydrogel induces specific immune tolerance to the graft cells.
  • a subject may be administered pancreatic islet cells to treat diabetes.
  • the subject may be administered pancreatic islet cells and a hydrogel engineered to display FasL (a "FasL hydrogel") in order to specific induce immune tolerance to the pancreatic islet cells.
  • FasL hydrogel a "FasL hydrogel”
  • the subject may be administered hepatocytes to treat acute liver failure or liver-based metabolic disorders.
  • the subject may be administered hepatocytes and a FasL hydrogel in order to induce immune tolerance to the hepatocytes.
  • the graft cell may be administered as a preparation of isolated cells or as part of a tissue or organ.
  • the graft cell may be allogenic.
  • the graft cell may be xenogenic.
  • the graft cell may be from a human, a non-human primate, a dog, a cat, a cow, a sheep, a horse, a rabbit, a mouse, or a rat.
  • the graft cell may be autologous or autogenic (from the subject being treated).
  • an autologous graft cell may be derived from autologous tissue by induced pluripotency and differentiation of the induced pluripotent cells to the desired autologous graft cell.
  • Cells from the subject may be used to induce immune tolerance to self that has been interrupted in autoimmune disease.
  • Exemplary cells suitable for use in these embodiments include mobilized hematopoietic stem cells, PBMCs, dendritic cells, and the like.
  • the cells may be chosen from those that naturally express self antigens that are targeted in the autoimmune disease.
  • type I diabetes is an autoimmune disease wherein the body reacts and rejects pancreatic islet ( ⁇ ) cells. In early stages of diabetes, before all islet cells are rejected, it can be possible to induce tolerance to islet cells and thereby prevent the progression of diabetes.
  • the subject may be in need of immune tolerance to a graft cell, and immune tolerance may be induced by administering a FasL hydrogel as described herein and the graft cell, and the graft cell is selected based on the condition to be treated.
  • the graft cell may be pancreatic islet cells.
  • the graft cell may be cells from the allograft donor, such as allograft bone marrow cells, allograft cardiac myocytes and allograft vascular cells, or other cells from the allograft donor as discussed above.
  • the graft cell may be cells from the xenograft donor, such as xenograft bone marrow cells, xenograft cardiac myocytes and xenograft vascular cells, or other cells from the xenograft donor as discussed above.
  • the graft cell may be autologous cells, such as cells derived from autologous tissue by induced pluripotency and differentiation of the induced pluripotent cells.
  • the subject may be in need of immune tolerance to a graft cell.
  • the graft cell may be selected from PBMCs, bone marrow cells, hematopoietic stem cells, stem cells, mesenchymal stem cells, dendritic cells, dendritic cells pulsed with autoantigens, human beta cell products, and splenocytes.
  • the graft cell may be pancreatic islet cells, or in addition or alternatively other cells as discussed above.
  • the graft cells may be cells from the allograft donor, such as cells selected from the group consisting of allograft bone marrow cells, allograft cardiac myocytes and allograft vascular cells, or other cells from the allograft donor as discussed above.
  • the graft cells may be cells from the xenograft donor, such as cells selected from the group consisting of xenograft bone marrow cells, xenograft cardiac myocytes and xenograft vascular cells, or other cells from the xenograft donor as discussed above.
  • the graft cells may be (i) a cell expressing the autoantigen (ii) a cell decorated with the autoantigen and (iii) a dendritic cell pulsed with the autoantigen.
  • the FasL biomaterial (such as a FasL hydrogel) and graft cell may be administered in the same composition, or may be administered separately.
  • the graft cell may be encapsulated by the FasL biomaterial.
  • the graft cell may be entrapped within the hydrogel or microgel biomaterial.
  • the FasL biomaterial (such as a FasL hydrogel) and graft cell may be administered to the same site in the subject, such as by local injection into approximately the same site.
  • the FasL biomaterial (such as a FasL hydrogel) and graft cell may be transplanted into the same site in the subject ( e.g., co-transplantation).
  • the methods may achieve long-term, specific immunosuppression at the site of the graft.
  • the administering is by transplantation.
  • Allogeneic islet graft acceptance may be achieved by simple co-transplantation of unmodified islets and FasL-presenting biomaterials without long term immunosuppression.
  • the FasL biomaterial (such as a FasL hydrogel) is administered with an immunosuppressive drug, such as rapamycin or any of the others mentioned above.
  • an immunosuppressive drug such as rapamycin or any of the others mentioned above.
  • the FasL biomaterial (such as a FasL hydrogel) and immunosuppressive drug may be formulated together (e.g., the hydrogel may comprise the immunosuppressive drug), or they may be administered in separate compositions, simultaneously or sequentially in any order. A shorter course of immunosuppressive drug may be required than when no FasL biomaterial is administered.
  • FasL biomaterials such as a FasL hydrogels
  • FasL biomaterials such as a FasL hydrogels
  • FasL hydrogels that comprise an immunosuppressive drug may provide controlled release of the drug within the graft microenvironment, or contain the graft in the form of a capsule engineered with these immunomodulatory molecules (FasL).
  • Administering a FasL biomaterial (such as a FasL hydrogel) as described herein with an immunosuppressive drug may achieve a synergistic immunosuppressive effect.
  • the immunosuppressive drug such as rapamycin
  • the immunosuppressive drug may work in synergy with FasL to specifically eliminate pathogenic T effector cells while expanding T regulatory cells, thereby tipping the balance of immune response towards protection.
  • this synergistic effect may be achieved by the FasL moiety activating death receptor-mediated extrinsic apoptosis in Teffector cells, while the immunosuppressive drug (such as rapamycin) activates mitochondria-mediated intrinsic apoptosis. See, e.g., Ju et al., Nature 373(6513): 444-448 (1995 ); and Yellen et al., Cell Cycle, 10(22): 3948-3956 (2011 ).
  • Administering a FasL biomaterial (such as a FasL hydrogel) as described herein with an immunosuppressive drug may not impair the systemic immune response, and may increase the ratio of T regulatory cells to T helper cells.
  • T regulatory cells play an important role in modulating immune responses and they the inflammatory cues and infiltrate into rejecting grafts.
  • the FasL biomaterial such as a FasL hydrogel
  • the FasL biomaterial may be administered in an amount effective to induce immunosuppression or induce specific immune tolerance.
  • Effective amounts of FasL will vary depending on the subject being treated, the route of administration, and the nature and severity of the condition to be treated.
  • the amounts of FasL used in the examples below are illustrative and can be converted to doses for other subject based on the following table:
  • an effective dose of FasL may be from less than about 0.2 ⁇ g/kg/day to at least about 10 ⁇ g/kg/day, or more, based on the FasL moiety.
  • methods described herein may be carried out using daily doses of FasL at amounts of less than about 0.2 ⁇ g/kg/day, about 0.2 ⁇ g/kg/day, about 0.5 ⁇ g/kg/day, about 1 ⁇ g/kg/day, about 1.5 ⁇ g/kg/day, about 2 ⁇ g/kg/day, about 2.5 ⁇ g/kg/day, about 3 ⁇ g/kg/day, about 3.5 ⁇ g/kg/day, about 4 ⁇ g/kg/day, about 4.5 ⁇ g/kg/day, about 5 ⁇ g/kg/day, or more.
  • Type 1 diabetes is an autoimmune disease characterized by loss of insulin-producing ⁇ -cell mass, and thereby glycemic control, due to a coordinated immune response against ⁇ -cell specific antigens requiring CD4 + T cells.
  • Restoration of ⁇ -cell mass through allogeneic islet transplantation is currently the preferred clinical intervention to improve glycemic control in patients with severe glycemic instability.
  • longevity of allogeneic grafts is limited not only by host immune responses, but also by secondary graft failure due to toxic effects of chronic immunosuppression required to control rejection.
  • Pathogenic T effector (Teff) cells are the major culprit of islet allograft destruction. Therefore, a promising strategy to increase the functional longevity of islet allografts without the need for long-term immunosuppression comprises novel therapies that target Teff cells for elimination, mitigating their pathogenic function.
  • T cells Upon activation, T cells upregulate Fas and become sensitive to FasL-mediated apoptosis, a process that plays a critical role in activation-induced cell death (AICD) and tolerance to self-antigens.
  • AICD activation-induced cell death
  • FasL results in massive lymphoproliferation and autoimmune pathologies in rodents and humans, demonstrating lack of compensatory mechanisms and the importance of this pathway for immune regulation. Recognizing the immunomodulatory potential of this pathway, several groups have successfully used FasL gene therapy to mitigate allogeneic immune responses for graft acceptance in experimental animal models. Although these interventions show efficacy, the unknown safety profile of sustained ectopic expression of FasL in target tissues, as well as technical and regulatory challenges of gene therapy, limit their clinical potential.
  • FasL only contributes to AICD in its membrane-bound, oligomeric form.
  • Matrix metalloproteinases MMP can cleave FasL into an extracellular soluble form that inhibits apoptosis and acts as a chemoattractant for neutrophils, accelerating the destruction of allografts.
  • Islet transplantation is a promising therapy for Type 1 diabetes.
  • chronic immunosuppression to control rejection of allogeneic islets induces morbidities and impairs islet function.
  • T-effector cells are responsible for islet allograft rejection and express Fas death receptor following activation, becoming sensitive to Fas-mediated apoptosis.
  • localized immunomodulation using microgels presenting an apoptotic form of Fas ligand (SA-FasL) as described herein results in prolonged survival of allogeneic islet grafts, as shown in diabetic mice.
  • SA-FasL-microgels a short course of rapamycin treatment can boost the immunomodulatory efficacy of SA-FasL-microgels, resulting in acceptance and function of all allografts over an extended period of time, such as 200 days in the experiment reported below.
  • treated subjects may exhibit normal systemic responses to donor antigens, implying immune privilege of the graft, and increased CD4 + CD25 + FoxP3 + T-regulatory cells in the graft and draining lymph nodes.
  • deletion of T-regulatory cells resulted in acute rejection of established islet allografts.
  • FasL-engineered biomaterials wherein streptavidin-conjugated FasL (SA-FasL) is displayed on a biocompatible material, that is a hydrogel, such as a polyethylene glycol (PEG) hydrogel.
  • SA-FasL streptavidin-conjugated FasL
  • PEG polyethylene glycol
  • the SA-FasL-engineered biomaterials are useful, for example, for immunomodulation, such as for preventing or reducing the risks of rejection of cellular or tissue grafts, such as for preventing or reducing the risks of foreign graft rejection, for preventing or reducing the risks of rejection of pancreatic islet transplantation, and/or for preventing or reducing the risks of rejection of stem cells, human pancreatic beta cell products (such as may be used for the treatment of type 1 diabetes), and in conjunction with other treatments and/or the treatment of other disorders that may benefit from cellular or tissue grafts.
  • immunomodulation such as for preventing or reducing the risks of rejection of cellular or tissue grafts, such as for preventing or reducing the risks of foreign graft rejection, for preventing or reducing the risks of rejection of pancreatic islet transplantation, and/or for preventing or reducing the risks of rejection of stem cells, human pancreatic beta cell products (such as may be used for the treatment of type 1 diabetes), and in conjunction with other treatments
  • the SA-FasL-engineered biomaterials described herein are useful in the treatment of autoimmune diseases, such as type I diabetes, the prevention of rejection of cellular and tissue grafts, such stem cells, pancreatic islets, hematopoietic stem cells, hepatocytes, mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, human beta cell products derived from stem cells, and in conjunction with the treatment of various hematopoietic and immune deficiency disorders through the use of stem cells.
  • autoimmune diseases such as type I diabetes
  • pancreatic islets such stem cells, pancreatic islets, hematopoietic stem cells, hepatocytes, mesenchymal stem cells, induced pluripotent stem cells, embryonic stem cells, human beta cell products derived from stem cells.
  • the streptavidin-conjugated FasL construct (SA-FasL) used in specific embodiments described herein has been described per se, and has been shown to prevent the rejection of allogeneic pancreatic islets under a short course of rapamycin treatment.
  • PEG hydrogels engineered to display SA-FasL protein on their surface have been shown to be effective in preventing the rejection of co-transplanted pancreatic islets when used in combination with a short course of immunosuppressive drug rapamycin in chemically diabetic mice.
  • SA-FasL has unique mechanisms of action that can be maximally exploited using hydrogels as a delivery vehicle. Given the critical role of FasL in self-tolerance and that there is no compensatory mechanisms when deficient, this molecule has advantages over other biologics and immunosuppressive drugs used to treat autoimmunity and graft rejection. For example, autoreactive and alloreactive T cells, when activated, upregulate the Fas receptor, and as such become the direct target of SA-FasL. Therefore, SA-FasL has the potential to specifically eliminate auto and alloreactive T cells, without the knowledge of the T cell repertoire for antigen specificity. This pathway has not been targeted for therapeutic purposes and as such it is unique.
  • this concept has the efficacy and specificity over the present technologies used by the industry for the treatment of autoimmunity and graft rejection, which are not only ineffective but also have various side effects, for example those associated with standard immunosuppression used for autoimmunity and graft rejection.
  • Biotinylated microgels can be produced by reacting biotin-(poly-ethylene-glycol (PEG))-thiol with maleimide-terminated 4-arm poly ethylene glycol (PEG-4MAL) macromer, and generating 150 ⁇ m diameter microgels crosslinked with dithiothreitol (DTT) via microfluidics polymerization ( FIG. 1A ).
  • the resulting microgels display covalently-tethered biotin capable of capturing streptavidin (SA) with high affinity ( FIG. 1B ).
  • Biotin-specific capture of SA on microgels varied linearly with concentration of SA in the tethering solution up to a saturating concentration of 150 ⁇ g/mL ( FIG.
  • rat islets were cultured with SA-FasL-presenting microgels (1:2 islet microgel ratio) for 24 hours. There were no differences between islets co-cultured with SA-FasL-presenting microgels and free islets (control) in metabolic activity ( FIG. 10A ), glucose-stimulated insulin secretion ( FIG. 10B ), live-dead staining ( FIG. 10D ), or insulin and glucagon expression patterns ( FIG. 10E ).
  • Example 2 Presenting SA-FasL on microgels prolongs local SA-FasL delivery at graft sites.
  • SA-FasL labelled with a near-infrared fluorescent dye
  • SA-FasL was immobilized on biotinylated microgels, which were implanted under the kidney capsule of mice.
  • Longitudinal tracking of labelled SA-FasL was performed on an in vivo imaging system for 21 days. Images for fluorescent signal show concentrated signal localized to the area around the kidneys in mice receiving labelled SA-FasL-presenting microgels, whereas the fluorescent signal is diffuse for mice receiving labelled free SA-FasL without the microgel carrier ( FIG. 2A ).
  • mice receiving only labelled SA-FasL without the microgel delivery vehicle, the protein was rapidly cleared from the transplant site, with a 60% reduction in signal by day 1 post-implantation and negligible signal by day 7 after implantation ( FIG. 2B ).
  • mice receiving SA-FasL-presenting microgels displayed significantly higher levels of SA-FasL over time with elevated levels comparable to day 0 signal at the site of implantation over 7 days post-transplantation.
  • Analysis of retention profiles using single exponential decay curve fits showed significantly longer retention times for SA-FasL- presenting microgels compared to free SA-FasL (half-life 3.0 ⁇ 0.8 days vs. 0.70 ⁇ 0.40 days, p ⁇ 0.0001 ) .
  • FIG. 11 shows that microgels at the implant site could clearly be observed at day 21 as determined by histology.
  • Example 3 Co-transplantation of allogenic islets and SA-FasL-engineered microgels restores long-term glycemic control without the use of chronic immunosuppression or modification of the islets.
  • microgel-based SA-FasL presentation avoidance of the chemical modification of islets, which may overcome a potential negative impact on islet viability and function, and also provide a better translatable strategy as an off-the-shelf product.
  • SA-FasL-engineered microgels restores long-term glycemic control without the use of chronic immunosuppression or islet modification.
  • Example 4 Recipients of SA-FasL-engineered microgels maintain systemic immune competence.
  • Unmodified microgels plus rapamycin group showed a trend towards increased numbers of Treg cells that reached significance in the graft-infiltrating lymphocytes on day 7 post-transplantation.
  • Mice receiving SA-FasL-engineered microgels and rapamycin had an increased ratio of Treg to CD4 + and CD8 + Teff cells in the graft ( p ⁇ 0.05 for Treg:CD8 + Teff) and graft draining LNs ( p ⁇ 0.05 for both Treg:Teff populations) compared to control mice receiving unmodified microgels alone or in combination with rapamycin ( FIG. 4B ).
  • Example 5 The SA-FasL-microgel strategy improves transplanted islet function without chronic immunosuppression in a clinically-relevant transplant site.
  • the kidney capsule is an experimentally convenient transplant site to study cell delivery, but it has limitations for clinical adoption. We therefore examined allogeneic islet transplantation into the murine epididymal fat pad.
  • the epididymal fat pad in mice is analogous to the omentum in humans.
  • the omentum represents a clinically relevant islet transplant site. See Baidal et al., N Engl J Med 376(19): 1887-1889 (2017 ); and Berman et al., Diabetes, 65(5): 1350-1361 (2016 ).
  • grafts were delivered within a protease-degradable PEG hydrogel with controlled VEGF release that improves islet engraftment.
  • Microgel synthesis and characterization A microgel precursor solution containing 5% w/v PEG-4MAL (20kDa, Laysan Bio) and 1.0 mM biotin-PEG-thiol (1 kDa, Nanocs) was reacted for 15 min in PBS. This precursor was dispersed into droplets and subsequently was crosslinked within mineral oil (Sigma) containing 2% SPAN80 (Sigma) and a 1:15 emulsion of 30 mg/mL dithiothreitol (Sigma) on a microfluidic chip, as described previously. See Headen et al., Advanced Materials, 26: 3003-3008 (2014 ).
  • Control microgels which did not contain biotin-PEG-thiol were also synthesized using this protocol. After washing microgels 5 times by centrifugation in 1% bovine serum albumin (Sigma) in PBS, 10 4 microgels were incubated with varying concentrations of a streptavidin-AlexaFluor488 conjugate for 30 min in 500 ⁇ L PBS, and were washed 5 times by centrifugation to remove unbound SA. Microgels from each sample were placed in a 96-well plate and fluorescence was measured on a plate reader (Perkin Elmer HTS 7000). Biotin and control microgels were also synthesized with a covalently bound peptide (GRGDSPC)-AlexaFluor594 conjugate for capsule visualization, and were fluorescently imaged to confirm biotin-specific SA immobilization.
  • GGSDSPC covalently bound peptide
  • a static glucose-stimulated insulin release (GSIR) assay was used to assess the insulin secretion of islets post co-culture, stimulating with low (3 mM) and high Krebs buffer (11 mM) for 1 h each. A second exposure to basal conditions was performed for an additional 1 h.
  • a rat insulin ELISA was used to quantify GSIR samples (Mercodia, Inc., Winston Salem, NC). Inflammatory cytokines from co-culture supernatant were analyzed via a multiplexing magnetic bead-based antibody detection kit (Milliplex Rat Cytokine Panel with IFNg, IL-1b, IL-6, IL-17A, MCP-1, MIP-1a) following the manufacturer's instructions.
  • Islet transplantation BALB/c pancreatic islets were isolated using Liberase TL as a digestive enzyme (Roche Life Science) and purified by a Ficoll density gradient as previously published. See Yolcu et al., Immunity 17: 795-808 (2002 ). To biotinylate islets, overnight cultured islets were incubated in 5 ⁇ M EZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific) for 30 min at room temperature, washed extensively with PBS to remove unbound biotin solution. Biotinylated islets and microgels were engineered with SA-FasL ( ⁇ 150 ng/500 islets and 1-10 ⁇ g/1000 microgels).
  • islets were co-transplanted with 1000 microgels into streptozotocin diabetic (200 mg/kg i.p., diabetes (> 250 mg/dL) confirmed on two consecutive days) C57BL/6 or B6.129(Cg)- Foxp3 tm3(DTR / GFP)Ayr /J (C57BL/6.FoxP3 EGFP/DTR ) recipients, where indicated.
  • streptozotocin diabetic 200 mg/kg i.p., diabetes (> 250 mg/dL) confirmed on two consecutive days) C57BL/6 or B6.129(Cg)- Foxp3 tm3(DTR / GFP)Ayr /J (C57BL/6.FoxP3 EGFP/DTR ) recipients, where indicated.
  • islet graft recipients were injected i.p. with diphtheria toxin (50 ⁇ g/kg body weight) and depletion was confirmed 3 days later in peripheral blood lymphocytes using flow cytometry.
  • Spleen, kidney, and kidney draining lymph nodes were harvested from rejecting and long-term mice (> 200 days). Single cells were prepared from the spleen and lymph nodes by gentle mechanical dispersion and from islet harboring kidney by collagenase digestion. Cells were stained using antibodies to cell surface markers (Alexa 700-CD4 Ab, APC-Cy7-CD8 Ab, PE-Cy7-CD25 Ab from Pharmingen, BD, and eFlour 450-CD44 Ab and PerCP-Cy5.5-CD62L Ab from eBioscience). Intracellular FoxP3 staining was carried out on fixed/permeablized cells using FoxP3 Transcription Factor Staining Buffer set (eBioscience). Data was collected using BD LSR II and analyzed using Diva software. Data was graphed using GraphPad Prism and Welch's t test was used to determine significance between groups, p ⁇ 0.05 was considered significant.
  • Splenocytes harvested from selected group of transplant recipients were labeled with CFSE and used as responders to irradiated (2000 cGy) splenocytes from donor or third party C3H mice in a standard in vitro proliferation assay. See E. S. Yolcu et al., J Immunol, 187: 5901-5909 (2011 ). After 4 days in culture, cells were stained with 7AAD and fluorescence-conjugated Abs against CD4 and CD8, and analyzed for CFSE dilution by gating on live cells using BD LSR II. Data was analyzed using Diva software. Data was graphed using GraphPad Prism and Welch's t test was used to determine significance between groups, p ⁇ 0.05 was considered significant.
  • Staining was performed using rabbit anti-glucagon mAb (Cell Signaling) and guinea pig anti-insulin polyclonal antibody (Dako) as primary antibodies, followed by washing and staining with AlexaFluor-647-conjuaged goat anti-rabbit antibody (Life Technologies) and AlexaFluor-555-conjugated anti-guinea pig antibody (Invitrogen).
  • Hoechst 33342 (Molecular Probes) was used to stain DNA. Fluorescent images were obtained using a Leica TCS SP5 confocal microscopy under 10X magnification.

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Claims (21)

  1. Biomatériau à modification à base de protéine FasL, dans lequel le biomatériau est un hydrogel, dans lequel l'hydrogel comprend une protéine FasL chimère comprenant une fraction de FasL et une fraction de streptavidine ou d'avidine conjuguée par le biais de la biotine à l'hydrogel.
  2. Biomatériau selon la revendication 1, dans lequel l'hydrogel est un microgel.
  3. Biomatériau selon les revendications 1 ou 2, dans lequel l'hydrogel est un microgel de polyéthylène glycol (PEG) modifié pour afficher une fraction de biotine.
  4. Biomatériau selon l'une quelconque des revendications 1 à 3, dans lequel la fraction de FasL est une protéine FasL résistante aux métalloprotéinases matricielles.
  5. Biomatériau selon la revendication 1, dans lequel le biomatériau comprend en outre un médicament immunosuppresseur.
  6. Biomatériau selon la revendication 5, dans lequel le médicament immunosuppresseur est la rapamycine.
  7. Biomatériau selon la revendication 1, dans lequel le biomatériau comprend en outre une cellule greffée.
  8. Biomatériau selon la revendication 7, dans lequel la cellule greffée est encapsulée par le biomatériau.
  9. Biomatériau selon la revendication 7, dans lequel la cellule greffée n'est pas encapsulée par le biomatériau.
  10. Biomatériau à modification à base de protéine FasL selon l'une quelconque des revendications 1 à 9 pour utilisation dans l'induction d'une tolérance immunitaire chez un sujet qui en a besoin.
  11. Biomatériau pour utilisation selon la revendication 10, dans lequel l'hydrogel est transplanté chez le sujet.
  12. Biomatériau pour utilisation selon la revendication 10 en combinaison avec un médicament immunosuppresseur, éventuellement dans lequel le médicament immunosuppresseur est la rapamycine.
  13. Biomatériau pour utilisation selon la revendication 10, dans lequel le sujet est un humain, un primate non humain, un cochon, un chien, un chat, une vache, un mouton, un cheval, un lapin, une souris ou un rat.
  14. Biomatériau pour utilisation selon la revendication 10, dans lequel le sujet a besoin d'une tolérance immunitaire vis-à-vis d'une cellule greffée, éventuellement dans lequel on administre au sujet une cellule greffée, éventuellement dans lequel le biomatériau comprend en outre la cellule greffée, éventuellement dans lequel la cellule greffée est encapsulée par le biomatériau, éventuellement dans lequel la cellule greffée est choisie parmi le groupe constitué de PBMC, de cellules de la moelle osseuse, de cellules souches hématopoïétiques, de cellules souches, de cellules souches mésenchymateuses, de cellules dendritiques, de cellules dendritiques pulsées avec des auto-antigènes, de produits de cellules bêta humaines et de splénocytes.
  15. Biomatériau pour utilisation selon la revendication 10, dans lequel le sujet a besoin d'une tolérance immunitaire vis-à-vis d'une cellule greffée, éventuellement dans lequel on administre au sujet une cellule greffée, éventuellement dans lequel la cellule greffée n'est pas encapsulée par le biomatériau, éventuellement dans lequel la cellule greffée est choisie parmi le groupe constitué de PBMC, de cellules de la moelle osseuse, de cellules souches hématopoïétiques, de cellules souches, de cellules souches mésenchymateuses, de cellules dendritiques, de cellules dendritiques pulsées avec des auto-antigènes, de produits de cellules bêta humaines et de splénocytes.
  16. Biomatériau pour utilisation selon la revendication 10, dans lequel le sujet a besoin d'un traitement pour le diabète de type 1, éventuellement dans lequel on administre au sujet des cellules d'îlots pancréatiques.
  17. Biomatériau pour utilisation selon la revendication 10, dans lequel le sujet a besoin d'un traitement ou d'une prévention contre le rejet d'allogreffe, éventuellement dans lequel on administre au sujet des cellules provenant d'un donneur d'allogreffe.
  18. Biomatériau pour utilisation selon la revendication 10, dans lequel le sujet a besoin d'un traitement ou d'une prévention contre le rejet de xénogreffe, éventuellement dans lequel on administre au sujet des cellules provenant d'un donneur de xénogreffe, éventuellement dans lequel le donneur de xénogreffe est un humain, un primate non humain, un cochon, un chien, un chat, une vache, un mouton, un cheval, un lapin, une souris ou un rat.
  19. Biomatériau pour utilisation selon la revendication 10, dans lequel le sujet a besoin d'un traitement ou d'une prévention contre le rejet d'autogreffe, éventuellement dans lequel on administre au sujet des cellules de greffe autologues, éventuellement dans lequel les cellules de greffe autologues sont obtenues par pluripotence induite.
  20. Biomatériau pour utilisation selon la revendication 10, dans lequel le sujet a besoin d'un traitement ou d'une prévention contre l'auto-immunité, éventuellement dans lequel on administre au sujet un auto-antigène présenté sur une cellule choisie parmi (i) une cellule exprimant l'auto-antigène (ii) une cellule décorée avec l'auto-antigène et (iii) une cellule dendritique puisée avec l'auto-antigène.
  21. Procédé de fabrication d'un biomatériau selon l'une quelconque des revendications 1 à 9, comprenant la mise en contact d'un biomatériau biotinylé avec une protéine FasL chimère comprenant une fraction de FasL et une fraction de streptavidine ou d'avidine.
EP18764623.7A 2017-03-10 2018-03-09 Biomatériaux à modification fasl ayant une fonction immunomodulatrice Active EP3592392B1 (fr)

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SM20230437T SMT202300437T1 (it) 2017-03-10 2018-03-09 Biomateriali ingegnerizzati con fasl con funzione immunomodulatoria
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WO2024182722A1 (fr) * 2023-03-01 2024-09-06 Georgia Tech Research Corporation Nanoparticules thérapeutiques pour l'acceptation immunitaire d'organe solide
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US7927602B2 (en) * 2002-07-23 2011-04-19 University Of Louisville Research Foundation, Inc. Fas ligand-avidin/streptavidin fusion proteins
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SI3592392T1 (sl) 2024-02-29
EP3592392A4 (fr) 2020-12-16
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EP4299749A3 (fr) 2024-03-27
EP3592392A1 (fr) 2020-01-15
US20200046780A1 (en) 2020-02-13
PT3592392T (pt) 2023-11-23
JP2020511531A (ja) 2020-04-16
HUE064306T2 (hu) 2024-02-28
US20230310510A1 (en) 2023-10-05
CN118697854A (zh) 2024-09-27
WO2018165547A1 (fr) 2018-09-13
SMT202300437T1 (it) 2024-01-10
CN110582560B (zh) 2024-06-14
CN110582560A (zh) 2019-12-17
US11602547B2 (en) 2023-03-14
EP4299749B1 (fr) 2025-07-16
JP7335821B2 (ja) 2023-08-30
RS64754B1 (sr) 2023-11-30
FI3592392T3 (fi) 2023-11-09
HRP20231297T1 (hr) 2024-02-02
EP4299749C0 (fr) 2025-07-16
EP4299749A2 (fr) 2024-01-03
AU2018230474A1 (en) 2019-10-31
ES2963331T3 (es) 2024-03-26
PL3592392T3 (pl) 2024-03-18
CA3055908A1 (fr) 2018-09-13
AU2018230474B2 (en) 2023-08-03
LT3592392T (lt) 2024-01-25

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